Abstract
The highly conserved branch point sequence (BPS) of UACUAAC in Saccharomyces cerevisiae is initially recognized by the branch point-binding protein (BBP). Using systematic evolution of ligands by exponential enrichment we have determined that yeast BBP binds the branch point sequence UACUAAC with highest affinity and prefers an additional adenosine downstream of the BPS. Furthermore, we also found that a stem-loop upstream of the BPS enhances binding both to an artificially designed RNA (30-fold effect) and to an RNA from a yeast intron (3-fold effect). The zinc knuckles of BBP are partially responsible for the enhanced binding to the stem-loop but do not appear to have a significant role in the binding of BBP to single-strand RNA substrates. C-terminal deletions of BBP reveal that the linker regions between the two zinc knuckles and between the N-terminal RNA binding domains (KH and QUA2 domains) and the first zinc knuckle are important for binding to RNA. The lack of involvement of the second highly conserved zinc knuckle in RNA binding suggests that this zinc knuckle plays a different role in RNA processing than enhancing the binding of BBP to the BPS.
Highlights
Pyrimidine), in which only the U and A are critical [1]
These results suggest that the branch point-binding protein (BBP) zinc knuckles are important for nonspecific binding to the phosphate backbone, whereas the K homology (KH) and QUA2 domains are responsible for sequence specific binding to the branch point sequence (BPS)
BBP Zinc Knuckle Linker Regions Help in RNA Binding, whereas the Second Zinc Knuckle Does Not—Previously, we showed that the region of BBP containing the zinc knuckles is necessary for high affinity binding to the BPS but that the zinc knuckle domains worked through nonspecific binding because they could be replaced with the short basic peptide (RS)7 which rescued the binding affinity
Summary
Pyrimidine), in which only the U and A are critical [1]. When discussing the yeast branch point-binding protein we will use the term BBP, when discussing the human protein we will use the term SF1, and when discussing both proteins we will use the term BBP/SF1. It has not been determined if the BPS is the high affinity site for the KH/QUA2 domains and if BBP binds additional RNA elements through the zinc knuckles.
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